Synergistic composition for the treatment of diabetes mellitus

ABSTRACT

A composition for the treatment of diabetes in a subject in need thereof, said composition—comprising Trigonelline of concentration ranging between 20 to 30%, amino acids of concentration ranging between 20 to 60%, and soluble fiber of concentration ranging between 10 to 60%, optionally along with pharmaceutically acceptable additives, a process thereof and also, a method of treating diabetes.

This application is a second divisional of U.S. Ser. No. 10/846,299,filed May 14, 2004, which is the non-provisional of U.S. Ser. No.60/470,742, filed May 14, 2003 and which application(s) are incorporatedherein by reference. A claim of priority to both, to the extentappropriate is made

FIELD OF THE PRESENT INVENTION

The present invention relates to a synergistic composition for thetreatment of diabetes mellitus, and also a process of preparing thesynergistic composition.

BACKGROUND AND PRIOR ART REFERENCES

Diabetes Mellitus is the most common endocrine disease. This disease ischaracterized by poor regulation of blood glucose levels in humanbeings. Blood glucose is the source of energy for basic cell functions.This glucose is driven to the cell by insulin, which is secreted by thepancreas. Diabetes Mellitus is caused by inadequate insulin secretion bythe pancreas or the resistance generated by insulin receptors to theinsulin. Therefore, this disease is characterized by a metabolicabnormality. Diabetes is a major metabolic disorder in which the bodydoes not produce or properly use insulin and is characterized byhyperglycemia, glycosuria, hyperlipidemia, negative nitrogen balance andsometimes ketonemia. Diabetes is one of the most common diseasesaffecting human population today.

In India, as per WHO reports, about 60 million people would be sufferingfrom diabetes by the year 2025. This would put India as the #1 countryin the world affected from this epidemic disorder. Diabetes among urbanIndians has increased from 11.8% in 1995 to 13.2% in 2000. This disorderstrikes people during the most productive stage of their lives. Based onrecent studies conducted and published, it appears that this disease iscaused more by lifestyle and food habits than genetic predisposition forthe disease. This is particularly true in urban areas wherein thelifestyle results in people exercising inadequately or tending to eatmore processed, ready-to-eat food. This in turn has led to obesity andlater, to the onset of diabetes.

Diabetes Mellitus is not curable. Presently, this disorder is managed bytaking popular drugs available in the marketplace. These drugs fall intothe following categories: -

-   i. Pancreatic stimulators:—This class of drugs helps to stimulate    the pancreas, leading to increased secretion of insulin. This    addresses the diabetes caused by inadequate insulin secretion.-   ii. Insulin sensitizers:—This category of drugs improves the cell's    sensitivity to the presence of insulin, thereby improving the uptake    of glucose into the cells, leading to better blood sugar control.-   iii. Insulin:—This is exogenously supplemented in the case of people    suffering from both type I and type II diabetes.

As mentioned earlier, diabetes is a lifestyle disease and cannot becured. The current therapies available therefore only offer a bloodsugar management mechanism. As diabetes is a chronic, long-durationdisease, these drugs need to be taken on a sustained basis. Currently,available synthetic drugs suffer from concomitant side effects causeddue to long duration of usage. Literature survey indicates thatcardiovascular mortality was higher in patients with oral hypoglycemicsthan in those treated with diet and exercise alone or with insulin.Sulphonylureas cause hypoglycemia as a side effect. Biguanides causelactic acidosis. Oral hypoglycemia drugs also cause GIT irritation,weight gain, hypertension, etc. On continuous and constant exertion, thediabetic person is also liable for pancreatic fatigue. In addition, itis also seen that many of the existing drugs available lead to drugresistance in patients with long durations of use.

As mentioned earlier, the long-term complications of diabetes are moredamaging. This is caused by spikes in blood sugar in patients during theday. Increased blood sugar even for short periods leads to glycosylationof Haemoglobin. And glycosylated Haemoglobin causes long-termirreversible damages to eyes, kidneys, nerves and blood vessels.

Complications of Diabetes: - A wide-spread pathological change isthickening of capillary basement membrane, increase in vessel wallmatrix and cellular proliferation resulting in vascular complicationslike lumen narrowing, early atherosclerosis, sclerosis of glomerularcapillaries, retinopathy, neuropathy and peripheral vascularinsufficiency. The level of glycosylated hemoglobin (HbAlc) is alsoincreased in diabetes and is taken as an index of protein glycoslyation.It reflects the state of glycaemia over the preceding 2-3 months. Assuch, there is no drug available for the treatment of diabeticcomplications.

Consequently, the need of the hour is to develop safe and efficaciousdrugs that can help in the management of blood sugar in diabetesmellitus patients. This drug should lend itself for long-term usewithout any side effects and without developing resistance.

Ayurveda:—Ayurvedic literature refers to the usefulness of many plantextracts in the treatment of diabetes mellitus. In general,pharmacological studies over the last five decades have not brought outadequate effectiveness of such blood glucose lowering drugs. However,physicians in India who practice Ayurveda, Unani, and Siddha useextracts of leaves, flowers, fruits, seeds, wood, bark, roots, or evenwhole plants of more than a hundred Indian medicinal plants for thetreatment of diabetes.

In Ayurveda, each herb has properties of curbing many disorders.Therefore, the crude extracts of herb have failed in offering clinicallysignificant results in any single disorder. Comprehensive studies on thecomponents of the herb that are responsible for certain indications needto be undertaken to obtain effective medications from this therapy.

Alternative Approach / Therapy for Control and Management ofDiabetes:—In recent years, efforts have been focused on increasing betacell mass in diabetes through induction of islet neogenesis orpancreatic regeneration. In this context, the role of herbal remedies ishighly significant. Ayurveda aims at treating the disease at the rootlevel. Hence, it would be worth exploring the action of selected pureingredients composition from herbs for their pancreatic viabilityenhancement and neogenesis potential.

Fenugreek is used to treat diabetes, migraines, allergies and elevatedcholesterol in traditional medicinal practices in India. As per theancient Indian practice of Ayurveda and Naturopathy, fenugreek seed istraditionally taken in a powdered form, or boiled with water, or as asprouted seed for the control of blood sugar. It must also be noted thatFenugreek in India is used as a culinary spice and as a medicinal herb.In general, the varieties that are used for medicinal purpose havesmaller grains, are dark brown in colour and bitter in taste whencompared to the varieties used for culinary purposes. At present, thereis no documented evidence of better medicinal property of the varietiesused for medicinal purposes. Also, it appears that the medicinal effectof control of blood sugar obtained with the consumption of fenugreekseed is widely varying and cannot be relied upon as a reliable agent forcontrol of blood sugar.

Clinical Studies:—Fenugreek seeds contain about 50% dietary fiber ifused as a defatted powder. Similar to Guar Gum, fibers present in theseeds may slow gastric transit time. Slowing the rate and extent ofglucose absorption in the gut is not likely to be the sole mechanism forhypoglycemic benefit. Sauvaire et al isolated an amino acid present infenugreek seeds (4 Hydroxyisoleucine) that increases glucose-inducedinsulin release. Fenugreek seeds contain the alkaloid trigonelline,which has a hypoglycemic effect.

The following human studies demonstrate the role of fenugreek in bothtype 1 and type 2 diabetes therapy. Madar et al (U.S. Pat. No.5,847,109) found that 17 of 21 patients with type 2 diabetes had areduction in 2 hour postprandial serum glucose averaging 30 mg/dLfollowing administration of 15 grams of ground fenugreek seed (these areapproximate figures interpreted from a chart; numeric data were notgiven). Four patients had no significant change in post-prandial glucosemeasurements.

Sharma et al (European J. of Clinic Nutr Volume 44, Pages 301, 306,1990) studied fenugreek in type 1 and type 2 patients using aninteresting mode of delivery. Fenugreek powder was treated to remove thefamiliar taste, divided into two equal doses and incorporated intochapati, a type of unleavened bread. A serving of chapati was given withlunch and dinner. In a crossover, placebo-controlled trial with 60 type2 patients, the treatment group received 12.5 mg defatted fenugreekpowder in chapatis at lunch and dinner with isocaloric diets for 24weeks. This trial found a significant decrease in the area under thecurve for blood glucose concentration of 40.6% in the treatment group. Adouble blind study was conducted using isocaloric diets with or withoutdefatted fenugreek powder in 10 patients with type 1 diabetes for two10-day study periods. Five of the patients received fenugreek during thefirst 10 days and the rest received it during the second period. Thefenugreek diet significantly reduced fasting serum glucose levels from271.8±4.2 mg/dL to 196.2±49.5 mg/dL. After ingestion of a glucose load,serum glucose levels in the treatment group were reduced and the areaunder the curve for blood glucose concentration decreased significantlyby 18.7%. Significant reductions in total cholesterol and triglycerideshave been documented with the use of fenugreek.

Lessly Fowden, Helen Empratt and Alfred Smith of the Department ofBotany and Microbiology, University College, Gower Street, London did avery detailed study of Fenugreek seed and identified an Amino Acid—4Hydroxy Isoleucine in fenugreek seed. They published their findings inPhytochemistry, 1973, volume 12, page 1707 to 1711 under the title “4Hydroxy Iso Leucine from seeds of Trigonella Foenum Graecum”. This paperexplains a detailed method for extraction & purification of 4 HydroxyIso Leucine from Fenugreek seeds. They have also explained a method foridentification of this amino acid. They suggested ethyl alcohol & waterextraction of ground seeds of amino acid followed by ion exchangeseparation. This crude extract as per their method was further purified& crystallized to give a pure compound of 4 Hydroxy Iso Leucine. Theirstructure elucidation was also explained in the above article.

In 1989, N. W. Alcock, David H, G Grocet et al of Department ofChemistry University of Warwick published a detailed paper inPhytochemistry Volume 23, page 1835 to page 1841 of 1989 titled“Stereochemistry for the 4 Hydroxy Iso Leucine from Trigonella FoenumGraecum”. They used an extraction process as used by Lessly Fowden toget a pure compound of 4 Hydroxy Iso Leucine. They elucidated thestructure correctly. U.S. Pat. No. 5,470,879 of 1995 title “Treatment ofNon-Insulin dependant Diabetes” by Sauvaire et al talk about 4 HydroxyIso Leucine for increasing insulin secretion in diabetic patients.Coincidently, the method of extraction and purification used in thepatent is same as the one published by Lessly Fowden in 1973 in theprior art. This patent, however, discloses certain properties of 4Hydroxy Iso Leucine in stimulation of the pancreas to increase insulinsecretion. This patent studies the effect of 4 Hydroxy Iso Leucine inanimals only. They have disclosed studies invitro in rat pancreas,invivo in experimental rats, experimental dogs and mice. There is nostudy done in patients suffering from diabetes mellitus.

Ingestion of Amino Acid:—Protein Mixture leads to a significant increasein the insulin secretion response to Type II Diabetes, Diabetes Care2003 26, 625-630 by Dr. Luc J. C. Van L Maastricht University,Netherlands. The insulin response to carbohydrate consumption with TypeII Diabetes is enhanced by administering free Amino Acid/Proteinmixture. The Researchers examine the insulinotropic capacity of amixture containing free Leucine and Protein in Diabetic Patients. Theyfind that compared with carbohydrate ingestion alone, co-ingestion ofthe amino acid protein mixture led to significant insulin secretoryresponse among Type II Diabetes. This is tripled as per the study.

U.S. Pat. No. 5,847,109 of 1998 title “Galactomannan products ofcompositions containing the same by Garti et al claim method forIsolation of Fenugreek Galactomanin & use as a nutraceuticals inreducing post—prandial glucose, insulin response & cholesterol levels.They have quoted results of a one-week study on 22 subjects for thisclaim. The quantity of fenugreek derived galactomanin administration tothem was 10 grams.

U.S. Pat. No. 5,997,877 of 1999 by Peter Chang describes a process forthe fractionation of Fenugreek seeds & extraction of the variousfractions such as soluble dietary fibre, deflavoured fenugreek seed,high protein fenugreek meal, etc. There is no reference to any diabetictreatment.

U.S. Pat. No. 6,495,175 of 2002 by G. B. Rao et al describes ‘a methodfor obtaining substantially pure fixed oil, Oleoresin & dietary fibrefrom fenugreek seeds. There is no mention of any diabetic treatment withany of these compounds.

The hypoglycemic effect of the alkaloid Trigonelline of fenugreek seedhas been explained in the publication ‘LANCET’ 1967, 2 [7529]1311-2title ‘Hypoglycemic effect of trygonelline by Mishkins KYJ, Joseph B,Suleman F G et al.

OBJECTS OF THE PRESENT INVENTION

The main object of the present invention is to develop a synergisticcomposition for treating diabetes.

Another main object of the present invention is to develop a process forthe preparation of a synergistic composition from fenugreek seeds fortreating diabetes.

Yet another object of the present invention is to develop a method oftreating diabetes, using the synergistic composition obtained fromfenugreek seeds.

Still another object of the present invention is to develop a method oftreating diabetes, using Trigonelline.

SUMMARY OF THE PRESENT INVENTION

The present invention relates to a synergistic composition for thetreatment of diabetes in a subject in need thereof, saidcomposition-comprising Trigonelline of concentration ranging between 20to 30%, amino acids of concentration ranging between 20 to 60%, andsoluble fibre of concentration ranging between 10 to 60%, optionallyalong with pharmaceutically acceptable additives, a process thereof andalso, a method of treating diabetes.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

Accordingly, the present invention relates to a synergistic compositionfor the treatment of diabetes in a subject in need thereof, saidcomposition-comprising Trigonelline of concentration ranging between 20to 30%, amino acids of concentration ranging between 20 to 60%, andsoluble fibre of concentration ranging between 10 to 60%, optionallyalong with pharmaceutically acceptable additives, a process thereof andalso, a method of treating diabetes.

In still another embodiment of the present invention, wherein theinvention relates to a synergistic composition for the treatment ofdiabetes in a subject in need thereof, said composition-comprisingTrigonelline of concentration ranging between 20 to 30%, amino acids ofconcentration ranging between 20 to 60%, and soluble fibre ofconcentration ranging between 10 to 60%, optionally along withpharmaceutically acceptable additives. In still another embodiment ofthe present invention, wherein the amino acids are selected from a groupcomprising L-arginine, Leucine, Isoleucine, and 4-Hydroxyisoleucine.

In still another embodiment of the present invention, wherein thesoluble fiber is galactomannan.

In still another embodiment of the present invention, wherein theadditives are extracted from the same fenugreek which comprises of agroup containing galactomannan.

In still another embodiment of the present invention, wherein thediabetes is Type II diabetes.

In still another embodiment of the present invention, wherein thecomposition is obtained from plant Fenugreek.

In still another embodiment of the present invention, wherein thecomposition is free of adverse effects.

In still another embodiment of the present invention, wherein theinvention relates to a process for the preparation of a synergisticcomposition from plant fenugreek comprising Trigonelline ofconcentration ranging between 20 to 30%, amino acids of concentrationranging between 20 to 60%, and soluble fibre of concentration rangingbetween 10 to 60%, optionally along with pharmaceutically acceptableadditives, said process consisting steps of:

-   -   flaking fenugreek seeds,    -   extracting the flaked seeds with hydro-alcohol,    -   concentrating the extract under vacuum to remove alcohol,    -   extracting the concentrate with n-hexane to remove fats and        lipids,    -   diluting the defatted concentrated,    -   filtration of the diluted extract to remove insolubles to obtain        resultant,    -   filtering the resultant through column chromatography to elute        amino-acids, and Trigonelline, and    -   adding fibre of fenugreek into the amino acids and Trigonelline        to obtain the synergistic composition.

In still another embodiment of the present invention, wherein theextraction is at temperature ranging between 20 to 70° C., preferably35° C.

In still another embodiment of the present invention, wherein the flakesare of size of about 15 mm.

In still another embodiment of the present invention, wherein hydroalcohol is isopropyl alcohol and water in equal ratio.

In still another embodiment of the present invention, wherein theextraction is for time duration ranging between 3 to 24 hours,preferably 12 hours.

In still another embodiment of the present invention, wherein the aminoacids are selected from a group comprising L-arginine, Leucine,Isoleucine, and 4-Hydroxyisoleucine.

In still another embodiment of the present invention, wherein the columnchromatography is Ion exchange chromatography.

In still another embodiment of the present invention, wherein the fibreis Galactomannan.

In still another embodiment of the present invention, wherein theinvention relates to a method of treating diabetes in a subject in needthereof, said method comprising step of administering pharmaceuticallyacceptable amount of composition comprising Trigonelline ofconcentration ranging between 20 to 30%, amino acids of concentrationranging between 20 to 60%, and soluble fibre of concentration rangingbetween 10 to 60%, optionally along with pharmaceutically acceptableadditives, to the subject.

In still another embodiment of the present invention, whereinadministration of the synergistic composition is at dosage rangingbetween 50 to 2000 mg/kg body weight.

In still another embodiment of the present invention, wherein methodshows hypoglycemic effect of about 50%.

In still another embodiment of the present invention, wherein the methodshows about 300% increase in the viability of the islets.

In still another embodiment of the present invention, wherein the methodmaintains the morphology of the pancreatic cells.

In still another embodiment of the present invention, wherein thesynergistic composition is administered orally.

In still another embodiment of the present invention, wherein thesynergistic composition acts at the pancreas through potassium channelmediated insulin secretion.

In still another embodiment of the present invention, wherein theinvention relates to a method of treating diabetes in a subject in needthereof, said method comprising step of administering pharmaceuticallyacceptable amount of Trigonelline, amino acids and galactomannan fibers,optionally along with pharmaceutically acceptable additives, to thesubject.

In still another embodiment of the present invention, wherein theadditives are extracted from the same fenugreek which comprises of agroup containing galactomannan.

In still another embodiment of the present invention, wherein thediabetes is type II diabetes.

In still another embodiment of the present invention, wherein thecomposition is free of adverse effects.

In still another embodiment of the present invention, wherein theinvention relates the administration of the synergistic composition isat dosage ranging between 50 to 2000 mg/kg body weight.

In still another embodiment of the present invention, wherein the methodshows hypoglycemic effect of about 35%.

In still another embodiment of the present invention, wherein the methodshows about 300% increase in the viability of the islets.

In still another embodiment of the present invention, wherein the methodmaintains the morphology of the pancreatic cells.

In still another embodiment of the present invention, whereinTrigonelline, amino acids and galactomannan fibers, is administeredorally.

In still another embodiment of the present invention, whereinTrigonelline, amino acids and galactomannan fibers act at the pancreasthrough potassium channel mediated insulin secretion.

The comparative results of fenugreek seed powder (whole), and thecomposition of the instant application are shown below to establishinventiveness of the application.

Material % decrease in glucose level Fenugreek seed powder 18%Composition of the instant Application 51%The aforementioned table clearly shows that the inventors have been ableto reach the right combination of selective components of the fenugreek.In addition, the right combination is further supported by theinventiveness in using appropriate concentration ranges of each of thethree components of the composition. This makes the invention both noveland inventive in nature.

Further, the activity analysis of all the components of fenugreek showedvery interesting data. The activity was restricted to only a couple ofcomponents. Also, synergy was observed by a selective combination ofthree components of the composition. The same is elaborated with thehelp of experimental data as given below.

Components of fenugreek Hypoglycemic activity (%) Volatile flavorcompounds 0 Fixed oils 0 Soluble fibers 0 Trigonelline 31  Saponin 0Amino acids   8% Composition of the instant application  51%The aforementioned results clearly establish synergy of the composition.The activity is much more that the mere additive effect of individualcomponents. Rather, it is very clearly reflected in the data that thenon-active components are bringing down the activity of the activecomponents. Therefore, elimination of the inactive components led tosignificant increase in the overall activity.

Apart from the synergy as observed in the invention, the compositionleads to increase in the pancreatic beta cell mass increase in asubject. There is no other data available in accordance to ourknowledge, wherein the beta cell mass increase is been attained. Thisprovides an indication of physiological significance of instantcomposition. In addition, there is an increase of about 300% in theviability of the islets. Thus further supplements the physiologicalchanges required to provide a holistic benefit to a subject in thereof.

Thus, the composition is not only effective in new cases of diabetesmellitus, but also, would be effective in management of patients withresistance to other generation 1, 2, and 3 drugs.

Therefore, the patentability requirements of novelty, inventiveness, andutility are satisfied.

The current method of management of blood sugar in diabetes mellitusdoes not lend itself for long-term use due to side effects. Long-termcomplications of Diabetes are also well known and there are no effectiveremedies for them. Therefore, there is always a need for development ofnew compounds for the management of blood sugar.

Fenugreek Seed is known to possess anticholesterol and antidiabeticactivities. However the compounds responsible for effective antidiabeticproperties have not been clearly elucidated and studied in detail inhuman subjects. Fenugreek Seed contains, among other, several compoundssuch as:

-   1. Host of Amino Acids-   2. Volatile flavor compounds-   3. Fixed oils-   4. Soluble fibers like galactomannan-   5. Trigonelline-   6. Various Saponins

Apparently some of these in combination are responsible for antidiabeticproperties and some others in combination are responsible for lipidlowing qualities.

Earlier studies cited in this patent refer to seed powder and seedfibers administered in large quantities to obtain some glycemic control.However, the level of control demonstrated is not good enough to offerclinically significant glycemic control, which can be taken advantage ofby subject suffering from type 2 diabetes.

An amino acid (4 Hydroxyisoleucine) found in the seed has a property toincrease insulin secretion in glucose mediated way. This has been testedextensively in animals. However there is no study done in human subjectssuffering from diabetes type 2 to understand the impact of this aminoacid in the management of this type 2 diabetes along with safety forlong-term administration.

It is known from published studies that certain amino acids like Leucineimproves insulin secretion response among type 2 diabetes. They have notassumed the role of mainstream medication as yet. Perhaps, this branchedchain amino acid (4 Hydroxyisoleucine) behaves in a manner similar toLeucine.

The present invention is about identification of individual compounds ofFenugreek seed that have beneficiary effect in the case of type 2diabetes. The present invention illustrates the method for extraction ofa compound from Fenugreek seed, resulting in a combination of certainmolecules that act in tandem and with synergy and leads to effectivecontrol of blood glucose in subjects suffering from type 2 diabetes. Thepresent invention explains the composition of matter of the extractderived from Fenugreek seed.

The varieties that have high biological activities in mice wereidentified and subjected to extraction to understand individualcompounds present. It is found that these varieties are rich in:

-   -   1. Amino acids    -   2. Trigonelline    -   3. Soluble Fiber galactomannan.

The amino acids found in this seeds are L-arginine, leucine, isoleucineand 4-Hydroxyisoleucine. It was decided to work with seed screened foroptimal activity. Thin layer chromatographic method was developed toscreen for amino acids and Trigonelline. A quantitative method wasdeveloped for checking soluble fiber.

It was decided to work with the seeds whose ratios were optimum, afterestablishing the amino acids content and Trigonelline content by TLCmethods.

The seeds were subjected to flaking and hydro alcohol extraction attemperatures ranging from 2° C. to 70° C. and alcohol compositionranging from 50% to 90% (the remaining being de-mineralized water) for aperiod ranging from 3 hours to 24 hours, preferably about 12 hours. Theclear, filtered extract is concentrated to a predefined volume so as toremove maximum alcohol and diluted to a predetermined volume with water.The resulting solution is subjected to fine filtration followed by Ionexchange chromatography. The process of known prior art of ion exchangechromatography was used except for the selection of the ion exchangeresin, which can bind both Trigonelline as well as amino acidscompletely and the resin selection was made in such a way that thecompounds eluted in the same medium without leaving any traces on theresin.

The elutes were again passed through another weak acid Cation exchangeresin in H⁺ form to selectively remove the metabolites and impuritiesformed during the isolation, concentration and elution process. Thispart exclusively removes the smelly cyclisation products of 4 HydroxyIsoleucine and the Keto-methyl 4 Hydroxy valeric acid.

The resultant elute contained only amino acids and Trigonelline. Theliquor obtained was spray dried on co-current equipment having a discatomizer with a speed of 30,000 rpm. The resultant powder was in a finegranular form, which can be readily formulated after standardization.

In the above-mentioned process, the ratio of the active biomolecules,which were present in the original form i.e. seed, was maintained intotality in the finished product. This was an important criterion forthe activity of the said composition.

The said formulation, after mixing with suitable carriers (preferablythe Fenugreek fiber as the carrier), was subjected to animal studies aswell as human studies on diabetic subjects having very high glycosylatedHaemoglobin values.

EXPERIMENT 1:—After screening the seeds for the presence of total aminoacids and Trigonelline using Thin Layer chromatography on a pre-coatedSilica gel TLC plate using n-Butanol:Acetic acid:water in the ratio of12:8:2 and initial scanning using uv at 254 nm for the presence ofTrigonelline. Ninhydrin colour development was used for total amino acidpresence.

The selected seeds in a quantity of 1 Kg were flaked in a flaker toexpose the inner core, resulting in flakes of average 15 mm in size.These flakes were than subjected to Hydro alcohol extraction using 6litres of Isopropyl alcohol:water mixture in the ratio of 50:50 at 35°C. for 12 hours. The resultant liquid (about 5500 ml) was concentratedto a final volume of 150 ml under vacuum at 45-50° C. This liquid wasextracted with 3×50 ml of n-Hexane to remove fats and lipids. Thedefatted concentrate was diluted with de-mineralized water to a finalvolume of 500 ml. This liquid was then subjected to fine filtrationthrough 200-mesh size screen to remove insolubles.

The filtered liquid was then passed through a glass column of 500 mmlength×25 mm diameter containing 100 ml of strong acid Cation exchangeresin in H⁺form, freshly regenerated with 600 ml of 3% HCL in water,followed by washing to neutral pH. After passing the liquid, the columnis washed with de-mineralized water to neutral pH. The loaded amino acidand Trigonelline were eluted out with 200 ml of 0.5 N ammonia solution.The ammonia liquid is circulated in the column until it attains a stablepH of about 8.0

The resultant solution was then passed through a glass column of 800 mmlength×25 ml diameter containing 200 ml of freshly regenerated weak acidCation resin in gel form. The eluent from this column is a colourless,neutral liquid having only compounds such as amino acids andTrigonelline present in the ratio as in the mother seed. This product isspray dried in the following conditions:—

Air flow: Co-current Inlet Temperature: 165° C. Outlet Temperature:  85°C. Atomizer revolution: 30,000 rpmThe resultant granule from the spray drying process was found to be freeflowing and suitable for formulation.

The resultant powder is screened in HPLC for amino acids byderivatization method using DNFB (Dinitro Flouro Benzene) andTrigonelline using UV. This contains total amino acids in the ratio of20 to 60%, Trigonelline from 20 to 30% and soluble fiber (Galoctomannen)10 to 60%.

EXPERIMENT 2:—A Spray dried extract of Fenugreek as explained inExperiment 1 is taken. This is filled into 00 size capsules after mixingwith an excipient. The excipient is the fiber extracted from fenugreekseed. The fiber is extracted from the waste of the fenugreek seed afterthe extraction of the compound containing total amino acids andTrigonelline. The extraction procedure for the fiber is as below:—

The waste of fenugreek seed is boiled with 5-10 times its volume ofde-mineralized water for 2 to 3 hours at a temperature of 70-80° C. Thisis subjected to coarse and fine filtration. The filtered liquid istreated with activated charcoal to remove the coloring matter. Thisliquid is vacuum concentrated to 1/3^(rd) its original volume. To thisis added an alcohol to precipitate the fiber. The precipitate isfiltered to obtain the fiber residue. The fiber residue is dried andground to a fine powder. The powdered fiber mainly contains the complexsugar from the seed in the form of Galactomannan.

The excipient and the active drug as extracted in Experiment No. 1 aremixed in such a way as to get a composition of total amino acids in theratio of 20 to 60%, Trigonelline from 20 to 30% and soluble fiber(Galoctomannen) 10 to 60%.

EXPERIMENT 3:—A dose response study for the test drug was conducted inhealthy wistar rats weighing about 100-150 Gms. 96 rats, which weredivided into 6 equal groups, were given the test drug in different dosesranging from 250 mg/kg, 500 mg/kg, 1000 mg/kg and 2000 mg/kg, with agroup kept as control rats not receiving the test drug. On the basis ofthis experiment, it was found that administration of a single dose oftest drug at 500 mg per Kg, 1 gm/kg and 2 gms/kg produced hypoglycemiceffect. The onset of action was 4 hours after administration for 500 mgper Kg, 3 hours after administration of 1 gm/kg and 1 hour after theadministration of 2 gms/kg of test drug. The duration of action was 4hours for 500 mg per Kg, 7 hours for 1 gm/kg of drug and 24 hours for 2gms/kg of the drug dose. This experiment demonstrated that the drug hada good hypoglycemic effect of about 20% at a dose of 1 gm/kg in the caseof healthy wistar rats. Also, administration of 1 gm/kg of test drug for15 days showed reduction in blood sugar level from 96.33 to 78 mgmpercentage. The blood lipid profile was not altered remarkably.

EXPERIMENT 4:—Sub-acute toxicity for this test drug was conducted inSwiss wistar rats of either sex having weights of 150-250 gms. 4 groupsof 8 animals each were classified, with 1 as control group, and theremaining were drug treated at dosage levels 1 gm/kg body weight, 2gm/kg of body weight and 4 gm/kg of body weight.

-   1. These groups were administered drugs for a period of 15 days    continuously and monitored for the following:—    -   Body weight    -   Food consumption-   2. At the end of the trial, they were sacrificed and the organs    liver, kidney, lungs, spleen and stomach were taken for    histopathalogy and organ weight checking.-   3. Tests like kidney function test and liver function tests were    performed in all the animals.-   4. Effects of the drug on hematology parameters such as hemoglobin,    RBC count, WBC count, Neutrophils, Lymphocyte, Eosinophils and    Erythrocite sedimentation rate were performed.-   5. On the basis of the above studies, it has been observed that the    net gain in body weight of drug treated animals was similar to that    of the control. The food consumption of the drug treated groups    compared well with the control group.-   6. The administration of the drug did not show any significant    difference in the hematology parameters as compared to the control    group.-   7. The drug at all 3 levels of administration did not cause any    kidney toxicity in rats.-   8. The liver function test indicated normal liver function.-   9. The organ weights did not show any variation and compared well    with the control group.-   10. Histopathology of the internal organs reveal no abnormality in    the liver, kidney, spleen, stomach and lungs. On the basis of this    toxicity study, it is concluded that the drug is safe in rats at the    doses administered.

EXPERIMENT 5:—16 mice were induced diabetes by the administration ofAlloxan. By conducting blood test on these animals, they were confirmedto have high blood sugar levels. After confirmation of diabeticinduction, they were treated with the test drug at the dose of 2 gm/kgof the body weight for a period of 21-26 days. This time period wasdecided by the reversal of the sugar content in the urine of the mouse.The sugar content of the urine was tested everyday. The mice testedpositive initially. By 21^(st)-26^(th) day, the blood sugar in the urinetested negative during treatment with the drug. At the end of thetreatment, the animals were sacrificed and their pancreas extracted forfurther studies. At this time, the blood samples were also taken forblood sugar check. The pancreas was subjected to large-scale isolationof islets by the standard procedure elucidated in the article “LargeScale Isolation of Islets “by Tissue Culture of Adult Mouse Pancreas byY. M. Shewade, M. Umrani and R. R. Bhonde as published inTransplantation Proceedings, 31, 1721-1723 (1999). Isolated islets werestudied to check their viability. On the basis of this experiment, itwas found that the blood sugar level of mice increased to 300-320 by the3^(rd) day after the administration of Alloxan. The control group, whichwas not given the drug, showed a blood sugar of 480 by the 15^(th) day.Many mice in this group died during the study. Whereas, the drug treatedanimals at the dose of 2 gm/kg of body weight showed a decrease in theblood sugar level to 210 mgms. After the isolation and tissue culture ofthe islets as per the procedure, it was observed that the viability ofthe islets increased from 22% to 81%. The observations are tabulatedbelow:—

RESULTS OF PANCREATIC CELL STUDIES ISLETS CONTROL ALLOXAN ALLOXAN + DRUGMorphology Intact well Distorted Intact well defined defined Number 1135± 75.2   130 ± 5.1  780 ± 16.3 Diameter 104.6 ± 5.72  — 92.7 ± 4.32 Area 12775 ± 1160.5 — 11275 ± 110.3  Viability 92.3 ± 1.3  22.1 ± 3.3 81.3 ±5.6  1. Islets are completely distorted and damaged in Alloxan groupMice. 2. Islets are mature, big and nearly opaque in control mice (NoAlloxan, No Drug). 3. Islet size is smaller in Alloxan and drug group.The islets are translucent indicating there is repair and neogenesis.

This experiment proves that continued administration of the drugreverses diabetes in the case of mice that have been induced diabeteswith alloxan. The drug is leading to improvement in viability of betacells. This mechanism of action may help in restoring viability ofdamaged pancreas of type 2 diabetes. The pancreatic viabilityenhancement and pancreatic neogenesis potential are demonstrated in thisexperiment.

EXPERIMENT 6:—The effect of the composition prepared as per Experiment 1& 2 as well as their individual components separated on columnchromatography were studied on normal Swiss Albino mice (male) in theweight range of 25-30 gms for GTT. The animals were fed in the followingmanner.

-   1) Administration of 2 gm/kg of glucose by Inter Peritonially.-   2) Administration of the drug (Orally):

Total amino acids 10 mg/kg Trigonelline 75 mg/kg Hypoglycemic agent(Glyburide) 10 mg/kg Composition derived from Experiments 1 & 2 30 mg/kg

-   3) Measurement of BSL at 0 minutes & 1 hour.-   4) Immediately after blood removal at 1 hour, the animals were    sacrificed by tail flicking and the pancreas was removed and    homogenized using tris buffer. After centrifugation, the homogenate    was characterized by thin layer chromatography and K⁺ Ions by    Biolyte analyser. Following are the observations:

ANIMAL STUDY ON MICE Identifi- cation BSL Mark Weight BSL at after onthe (in 0 hrs 1 hour K⁺ Ions Description Animal grams) Sex (mg/dl)(mg/dl) (μMol) Normal FH 29 Male 398 239 27.6 Glyburide HB 29 Male 365251 22.5 Total Amino HBT 28 Male 372 340 19.8 Acids Trigonelline BT 28Male 341 233 21.8 Drug Blank 28 Male 404 200 18.3 Composition (Derivedfrom Expts. 1 & 2)

The above experiment clearly indicates the enhanced activity of thecomposition made by the experiments as compared to the individualbio-actives. This demonstrates synergy of action in the composition.This experiment also demonstrates a possible mechanism of action for thecomposition.

Potassium Channels

Ion channels consist of protein molecules designed to form water filledpores that cover the membrane and they can switch between open andclosed states. Ion channels are selective and have gating propertiesi.e., the mechanism that controls the transition between open and closedstates of the channel.

Many drugs act through ions. Oral antidiabetic drugs like sulphonylureastimulate release of insulin from pancreas. They act on receptors onpancreatic β cell membrane. As a result of combination of sulphonylureawith receptors, there is depolarization, which means reversal change intransmembrane potential of cell by reducing conductance of ATP sensitivek⁺channels. This enhances ca2+ influx, producing degranulation of β cellof pancreas.

Thereby increasing rate of insulin secretion at any glucoseconcentration.

Decreased K+ ion concentration indicates the repolarisation effect asthe ATP dependant I_(K-ATP)

EXPERIMENT 7:—A prospective uncontrolled Pilot study to assess efficacyand safety of (TEST DRUG) composition derived from Experiments 1 & 2 inpatients with Type II Diabetes Mellitus on treatment with Insulin. Thistype of patients were selected due to the viability enhancement/neogenesis potential exhibited in mice studies

Objectives:

The primary objectives of the study were to assess effect of TEST DRUGon fasting blood sugar levels (BSL) of Type II diabetic patients, whowere on treatment with insulin and to assess the safety. Secondaryobjectives were to check effects of TEST DRUG on post-prandial BSL,glycosylated Hb to verify whether the dosage of insulin can be reducedafter administration of TEST DRUG.

Study Design:

This clinical trial was a prospective, uncontrolled, multi centeredstudy.

Study Population:

-   -   30 patients    -   Type II diabetes mellitus    -   Fasting BSL between 100-140 mg/dl    -   Taking insulin for at least last 6 months    -   Stable BSL for last 8 weeks    -   Stable dose of insulin for last 8 weeks

Methodology:

Patients were screened for eligibility at −2 and 0 weeks. Eligiblepatients received TEST DRUG capsules. Treatment period was 24 weeks.Clinical and laboratory evaluation was done at 0, +2, +4, +6, +8, +12+16 and +24 weeks.

Treatment Details:

-   -   Product: TEST DRUG    -   Pharmaceutical form: Capsule    -   Subjects were asked to take 6 capsules per day in 3 divided        doses. Each capsule weighing 700mg

Criteria for Evaluation of Efficacy:

Efficacy of TEST DRUG was assessed on the basis of reduction in fastingand PP BSL, glycosylated Hb, number of hypoglycemic attack between twoconsecutive visits, insulin dose after administration of the test drugcompared to base line levels.

Criteria for Evaluation of Safety:

Safety criteria included adverse events, laboratory parameters, vitalsigns, physical examination and ECG examination

Statistical Method:

Mean blood sugar levels of at different visit were compared withbaseline by using Paired t test.

Efficacy Results:

-   1. Fasting blood sugar and post prandial blood sugar remained stable    even after reduction in Insulin administered-   2. Glycosylated Hb levels at V4 (Visits 4 week), V6 and V8 were    compared with that of the base line. Statistically significant    reduction was observed at V6 and V8-   3. Mean requirements of insulin (in terms of units consumed per day)    at various visits were compared with that of the base line.    Statistically significant reduction in requirement of amount of    insulin was observed at all visits.-   4. Hypoglycemic attacks between two successive visits were compared    with that of first two base line visits. Statistically significant    increase in number of hypoglycemic attacks was observed at V3 and    onwards.

Safety Results: There was no serious adverse event.

Based on this study for 6 month, it is proved that the drug is able tocontrol blood glucose level very effectively in type 2 diabetes patientswho do not respond to oral hypoglycemic agents. This drug is alsoeffective in controlling glycosilated haemoglobin content in type 2diabetes patients This indicates effective control in managing long termcomplications of diabetes.

The composition has a property of increasing the beta cell mass andstimulating the beta cell to secrete insulin, thereby increasing theviability of beta cells and enhancing the activity of diseased pancreas.

The composition has a property of synergistically working together tobetter control blood sugar as compared to the effect of individualcomponents.

The composition acts at the pancreas through potassium channel mediatedinsulin secretion

The composition controls the fasting blood sugar in type 2 diabetespatients who do not respond to oral hypoglycemic agents and who arestabilized on insulin

The composition controls the post prandial blood sugar in type 2diabetes patients who do not respond to oral hypoglycemic agents and whoare stabilized on insulin

The composition creates reduction in the dose of insulin in type 2diabetes patients who do not respond to oral hypoglycemic agents and whoare stabilized on insulin

The composition controls the glycosylated haemoglobin in type 2 diabetespatients who do not respond to oral hypoglycemic agents and who arestabilized on insulin

The composition with its properties of glycosilated haemoglobin, fastingblood sugar, post prandial blood sugar, pancreatic beta cell massenhancement and beta cell viability enhancement leads to better controlof blood sugar and thereby delays the onset of long-term diabeticcomplications of retinopathy, nephropathy and neuropathy in type 2diabetes patients who do not respond to oral hypoglycemic agents and whoare stabilized on insulin

1. A process for preparing a composition, the composition comprising:trigonelline of concentration ranging between 20 to 30% weight/weight(w/w), amino acids of concentration ranging between 20 to 60% w/w, andsoluble fiber of concentration ranging between 10 to 60% w/w, optionallyalong with pharmaceutically acceptable additives, the process consistingsteps of: a. flaking fenugreek (Trigonella foenum-graecum) seeds toobtain flaked seeds, b. extracting the flaked seeds with hydro-alcoholto obtain an extract, c. concentrating the extract under vacuum toremove alcohol to obtain a concentrate, d. extracting the concentratewith n-hexane to remove fats and lipids to obtain a defattedconcentrate, e. diluting the defatted concentrate to obtain a dilutedextract, f. filtering of the diluted extract to remove insolubles toobtain a resultant, g. subjecting the resultant to column chromatographywherein amino-acids, and trigonelline are eluted, and h. adding fiber offenugreek into the amino acids and trigonelline to obtain thecomposition.
 2. A process as claimed in claim 1, wherein the flakedseeds are extracted at a temperature ranging between 20 to 70° C.
 3. Aprocess as claimed in claim 1, wherein the flaked seeds are of size ofabout 15 millimeter(mm).
 4. A process as claimed in claim 1, wherein thehydro-alcohol is isopropyl alcohol and water in equal ratio.
 5. Aprocess as claimed in claim 1, wherein the flaked seeds are extractedfor a time duration ranging between 3 to 24 hours.
 6. A process asclaimed in claim 1, wherein the amino acids are selected from the groupconsisting of 1-arginine, leucine, isoleucine, and 4-hydroxyisoleucine.7. A process as claimed in claim 1, wherein the column chromatography ision exchange chromatography.
 8. A process as claimed in claim 1, whereinthe fiber is galactomannan.
 9. A process as claimed in claim 2, whereinthe flaked seeds are extracted at a temperature of 35° C.
 10. A processas claimed in claim 5, wherein the flaked seeds are extracted for a timeduration of 12 hours.